The invention relates to a method comprising synthesis and purification of mono- and di-derivatives of cyclodextrins (abbreviated to CD), also the synthesis of supports from these cyclodextrin derivatives. These novel supports comprise one or two spacer arms regioselectively bonded to the 2, 3 or 6 position of a glucoside unit of the cyclodextrin.
The invention also relates to the use of these supports for the preparation or separation of enantiomers, for asymmetric synthesis, for catalysis, for the preparation or separation of geometrical isomers or positional isomers or for the preparation or separation of organic molecules with a hydrophobic nature.
The invention also relates to a method comprising synthesis of polymers obtained from mono- and di-derivatives of cyclodextrins, also to the synthesis of supports from these cyclodextrin polymers.
The invention also relates to the use of the supports for the preparation or separation of enantiomers, for asymmetric synthesis, for catalysis, for the preparation or separation of geometrical isomers, or for the preparation or separation of organic molecules with a hydrophobic nature.
Separating organic molecules using the encapsulation properties of cyclodextrins has been carried out for a large number of years. See in particular M. L. Bender: xe2x80x9cCyclodextrin Chemistryxe2x80x9dxe2x80x94Springer Verlagxe2x80x94New York, 1978; R. J. Clarke: xe2x80x9cAdvanced Carbohydrates Chemistry and Biochemistryxe2x80x9d, 1988, 46, 205; W. Saenger: ANGEW CHEM.xe2x80x94Int. Edit., 1980, 19, 344; G. Wenz: ANGEW CHEM.xe2x80x94Int. Edit., 1996, 33, 803. These properties are currently widely exploited on an industrial scale in the perfume and flavouring industries and in the pharmaceutical industry. See in particular J. Szejtli: MED. CHEM. REV. 1994, 14, 353, and D. Duchene: J. COORD. CHEM., 1992, 21, 223.
The use of cyclodextrins bonded to supports has demonstrated their ability to separate and prepare organic molecules and positional isomers (see in particular Y. Kawaguchi: ANAL. CHEM. 1983, 55, 1852-1857, and K. Fujimura: ANAL. CHEM. 1983, 55, 446-450) or geometrical isomers (E/Z isomerism)xe2x80x94(see in particular Y. Inoue: J. AM. CHEM. SOC. 1995, 117, 11033-11034).
More recently, the same supports have been used to separate or prepare enantiomers (see in particular K. Takahaschi: J. INCL. PHENOM., 1994, 17, 1) or a variety of molecules by catalysis (see in particular G. Wenz: ANGEW. CHEM.xe2x80x94Int. Edit., 1996, 33, 803). This field has been expanding for about twenty years, both on the analytical and on a preparative level. This is particularly true in the pharmaceutical industry, where the health authorities of industrialised countries require the separate study of optical isomers of any chiral compound used in a medicament composition.
Native or derivative cyclodextrins have been the subject of a number of studies and cyclodextrins, bonded or otherwise to supports, are commercially available.
Supports based on cyclodextrin derivatives or based on polymers from these derivatives have not been chemically defined and are in the form of mixtures of mono- and poly-derivatives. Cyclodextrins contain a large number of hydroxyl functions of almost equivalent reactivity and up until now, chemically and regioselectively defined CD derivatives produced on an industrial scale have not existed.
The present invention provides access to mono- and di-derivatives of pure and regioselectively defined cyclodextrins as regards the position of the derivative on the glucoside unit: the 2, 3 or 6 position.
The derivatives can be grafted onto a mineral or organic matrix via a covalent hydrocarbon bond carrying a thioether function. The ensemble constitutes a support with increased selectivity over known supports for the following applications:
separation or preparation of organic molecules with a hydrophobic nature;
separation or preparation of positional isomers or geometrical isomers;
separation or preparation of enantiomers; or
asymmetric synthesis of chiral molecules.
The increased selectivity of these supports is an important factor for enabling them to be used on a laboratory, pilot or industrial scale, as it can reduce production costs.
The supports of the invention, which are completely original, can produce chromatographic performances which are hitherto unknown, in particular in the field of separation of enantiomers by liquid chromatography: the selectivities obtained prove to be higher than those currently obtained with commercially available cyclodextrin-based chiral columns.
A number of parameters combine to produce this unexpected result:
The regioselectivity of the bond between the glucoside unit of the cyclodextrin and the spacer arm connecting it to the organic or mineral support. The synthesis technique and purification of the cyclodextrin mono-derivative can produce practically pure derivatives bonded in the 2, 3 or 6 position of the glucoside unit of the cyclodextrin.
Synthesis of a support using a single spacer arm between the very high purity cyclodextrin mono-derivative and the functionalised silica gel.
The presence of a supplemental interaction side constituted by the thioether, sulphoxide or sulphone function on the spacer arm, enabling the creation of Van Der Waals type bonds with the solute, with the latter being engaged in other reactions with the cyclodextrin.
The combination of these interactions leads to the supports of the invention, which have a higher discriminating power over those described in the prior art.
The state of the art is represented by European patent application EP-A-0 608 703 and United States patent U.S. Pat. No. 4,539,399, which describes chromatographic supports based on cyclodextrins. Those supports are not chemically defined as their method of synthesis leads to undifferentiated mixtures of compounds mono- and poly-substituted in the three positions (2, 3 and 6) of the glucoside unit. Chromatographic supports using a spacer arm containing a thioether function have been widely used to separate enantiomers. As an example, Rosini et al. described the immobilisation of cinchona bark alkaloids with that type of arm in TETRAHEDRON LETT. 26, 3361-3364, 1985. More recently, Tambute et al. described the immobilisation of tyrosine derivatives using the same technique in NEW J. CHEM. 13, 625-637, 1989. More recently still, Caude et al. disclosed the results of their studies and have demonstrated the advantage of the thioether arm in terms of chemical stability in J. CHROMATOGR. 550, 357-382, 1991.
Salvadori et al. have shown the efficacy of quinine derivatives in the form of osmium tetraoxide adducts for the oxidation of asymmetric olefins in homogeneous and heterogeneous phases, in CHIRALITY 4, 43-49, 1992. Such derivatives were present in the form of copolymers of acrylonitrile and quinine derivatives carrying a sulphoxide spacer arm. The efficacy of the presence of the sulphoxide function was not discussed in that type of support used for asymmetrical synthesis.
Further, a route to mono-alkenylcyclodextrins has been described by Hanssian et al. in J. ORG. CHEM. 1995, 60, 4786-4797. They described the synthesis of mono-2-allyl-xcex1-cyclodextrin by the action of allyl bromide and lithium hydride in the presence of dimethylsulphoxide. The reaction medium was then purified with acetone, then chromatographic purification was carried out on virgin silica gel in a 90/10 v/v, then in a 40/10 v/v, acetonitrile/water mixture to obtain a monoallyl-xcex1-cyclodextrin. However, the authors admit, the data obtained from proton nuclear magnetic resonance showed the presence of 20% of mono-6-allyl-xcex1-cyclodextrin.
Schxc3xcrig et al (J. HIGH RESOLUT. CHROMATOGR. 13, 713-717, 1990) have described the synthesis of allyl, pentenyl and octenyl derivatives of xcex2-CD which were then grafted onto hydrogenopolysiloxanes (methylhydrogenopolysiloxane+dimethylpolysiloxane=copolymer) by hydrosilylation in toluene in the presence of dihydrogenoplatinum hexachloride. No structural study on the chemical purity and regioselectivity of the CD derivatives could confirm the 6 position as the attachment point for the alkenyl moieties. This 6 regioselectivity has been contested by Ciucanu and Konig in J. CHROMATOGR. A, 685, 166-171, 1994. These latter also described the synthesis and purification of permethyl- and perpropylmono-O-pent-1-enyl-xcex2-cyclodextrin, then hydrosilylation of the double bond with dimethoxymethylhydrosilane or dichloromethylhydrosilane.
Yi et al. (J. CHROM. A, 673, 219-230, 1994) described the synthesis of 4-allyloxy benzoyl type mono-derivatives in the 2 or 3 position, the remainder of the hydroxyl groups having been permethylated. However, the field of the nuclear magnetic resonance apparatus used for such compounds (200 MHz) appears to have been a little weak for complete identification of the products obtained, because of the complexity of the problem to be solved and the precision required for integrating the ethylene signals. Those derivatives were grafted onto hydromethylpolysiloxanes (copolymer of octamethylcyclotetrasiloxane and dimethoxyditolylsilane).
K. Fujita describes, in J. Am. CHEM. SOC., 108, 2030-2034, 1986, the synthesis and purification of mono- and 3A-3C and 3A-3D-di-O-(xcex2-naphthalene-sulphonyl)-xcex2-cyclodextrin.
Yi et al. described, in J. HETEROCYCLIC CHEM. 32, 621, 1995, different routes to type 6A-6C and 6A-6D-di-O-(4-alkoxyphenyl) di-derivatives which are completely permethylated. The regioselectivity of the compounds obtained was controlled by using ditosylates of differing sizes. The compounds were copolymerised with a dihydrodioctyldecamethylhexasiloxane.
Bradshaw et al. described the synthesis of di-derivatives of type 6A-6B-di-O-(4-allyloxyphenyl) per-O-methyl-xcex2-cyclodextrin type cyclodextrins using 2,4-dimethoxy-1,5-benzenedisulphonyldichloride. The derivatives were then copolymerised with a dihydrogenododecamethylhexasiloxane. See in particular ANAL. CHEM. 67, 23, 4437-4439, 1995.
Thuaud et al. described the synthesis of polymers containing cyclodextrin units by condensing the latter with bifunctional reactants (epichlorhydrin) (J. CHROMATOGR. 555, 53-64, 1991 and CHROMATOGRAPHIA, 36, 373-380, 1993). The structure of the polymer was not regioselectively defined.
That polymer has previously been synthesised by P. Sugiura et al. (BULL. CHEM. SOC. JPN. 62, 1643-1651, 1989) who had also used diepoxides as polymerising agents.
Polymerisation of ethylenic monomers has been carried out for a great many years (xe2x80x9cPrinciples of Polymer Chemistryxe2x80x9d, Paul J. Flory, Editor, Cornell Press New York, 1953 edition). Homopolymerisation of mono-derivatives and di-derivatives of cyclodextrin containing a polymerisable carbonxe2x80x94carbon double bond has not been described before the present invention. It is carried out using a free radical initiator (an azo or peroxide type compound) at a temperature of 50xc2x0 C. to 200xc2x0 C., the preferred temperature range being 100xc2x0 C. to 150xc2x0 C. The reaction periods are from 1 to 48 hours. Preferred solvents are toluene, dioxane, chloroform, tetrahydrofuran, alcohols, dimethylformamide, dimethylsulphoxide or a mixture of these solvents.
The polymerisation reaction may or may not be carried out in the presence of a support, the latter preferably being surface modified with ethylenic functions, hydrogenosilanes or thiols.
Copolymerisation of ethylenic monomers has also been widely described, for example in xe2x80x9cPrinciples of Polymer Chemistryxe2x80x9d cited above. The synthesis conditions are identical to those used for homopolymerisation (described above). The comonomers used can be monofunctional (for example styrene), bifunctional (for example divinylbenzene, ethanediol or tetramethyldisiloxane) or polyfunction (for example glycerol trimethacrylate).
The copolymerisation reaction may or may not be carried out in the presence of a support, the latter preferably being surface modified by ethylene, hydrogenosilane or thiol functions. Polymerisation by hydrosilylation is known per se and described in J. CHROMATOGR., 594, 283-290, 1992. The basic technique described in that article can be used to prepare cyclodextrin polymers. The reaction is preferably carried out in the presence of a catalyst, generally a metal complex, for example a platinum or rhodium complex, at temperatures of 50xc2x0 C. to 180xc2x0 C., preferably about 100xc2x0 C. Solvents which are inert to the polymerisation reaction taking place are used to dilute the reaction medium. Preferred solvents are toluene, dioxane, chloroform, tetrahydrofuran and xylene or mixtures of these solvents.
The reaction times are 1 to 48 hours as the kinetics of the hydrolysilylation polymerisation reaction are relatively slow.
The polymerisation reaction may or may not be carried out in the presence of a support, the latter preferably being surface modified by styryl, methacryloyl, methacrylamido, acrylamido, hydrogenosilane, vinyl or thiol functions.
Cyclodextrins are cyclic oligosaccharides with general formula: 
The value n=6 corresponds to an xcex1-cyclodextrin; the value n=7 corresponds to a xcex2-cyclodextrin; the value n=8 corresponds to a xcex3-cyclodextrin.
The alcohol functions can readily be transformed by a variety of groups such as acid chlorides and isocyanates, to produce esters or carbamates. Reaction with halides produces ethers. Whatever the reaction conditions used in the reactions, they always result in mixtures. Usually, a number of osidic units are concerned in the reactions described above with a regioselectivity (2, 3 or 6 position on the osidic unit) which is difficult to control.
The present invention describes a method for synthesis and purification of pure mono- and di-derivatives of cyclodextrin regioselectively bonded in the 2, 3 or 6 position of an osidic unit. Cyclodextrin di-derivatives may concern two different osidic units. The following general terminology is thus used: 
Pure cyclodextrins with formulae (Xa), (Xb), (Xc), (XIa), (XIb), (XIc), (XId), (XIe) and (XIf) can be obtained using the following reaction scheme: 
and pure (XIa), (XIb), (XIc), (XId), (XIe) and (XIf).
The reaction between cyclodextrin and the compound with general formula Txe2x80x94R4xe2x80x94CHxe2x95x90CH2 can be carried out in solvents usually used in organic chemistry, such as water, dioxane, tetrahydrofuran, toluene, halogenated solvents (chloroform, methylene chloride . . . ), ketones (acetone, methylethylketone), acetonitrile, dimethylformamide, dimethylsulphoxide or mixtures of these solvents.
For reasons of solubility of the starting product and the products formed, preferred solvents are dimethylsulphoxide and dimethylformamide. The low reactivity of the primary and secondary hydroxyl groups of cyclodextrins necessitates the use of large excesses of Txe2x80x94R4xe2x80x94CHxe2x95x90CH2. The reaction temperature is 0xc2x0 C. to 100xc2x0 C., preferably between 0xc2x0 C. to 30xc2x0 C., to encourage the formation of mono- and di-derivatives and prevent the formation of polyderivatives. Reaction times are 1 to 10 days. After reaction, the reaction medium is generally poured onto acetone to precipitate the different constituents of the reaction medium and isolate them by filtering.
The precipitate is purified by preparative chromatography on a silica or alumina gel, or zirconia or titanium oxide, or on an organic polymer type support, such as styrene-divinylbenzene or polyvinyl alcohol.
These supports are surface modified by amino functions (for example aminopropyl), alkyl functions (for example octyl or octadecyl), aryl functions (for example phenyl), or diol functions.
The chromatographic procedure is carried out using water-soluble organic solvents such as acetonitrile, ethanol, methanol, isopropanol, dioxane, dimethylsulphoxide and dimethylformamide, mixed with water, at temperatures of 0xc2x0 C. to 80xc2x0 C., preferably 15xc2x0 C. to 30xc2x0 C.
Separation is monitored by refractive index detection and the purity of the cyclodextrin derivatives is determined by HPLC or TLC.
HPLC is carried out on a 100 xc3x85, 5 xcexcm aminopropyl silica column with dimensions of 250xc3x974.6 mm, RI detection, eluting with acetonitrile/water, 70/30 by volume. The TLC system uses virgin xe2x80x9cMerckxe2x80x9d silica plates. The mobile phase is a 50/50 by volume mixture of 30% ammonia and ethanol. Iodine vapour is used to reveal.
The purified products are isolated by vacuum evaporating the water and organic solvent at a temperature of 40xc2x0 C. to 80xc2x0 C. bulk in a vacuum varying from 1 to 50 mm of mercury.
The chemical balance of the reaction is as follows: 
where m is 1 to 24 (moles/mole of CD).
Formula (X) represents the possible mono-O-alkenyl-CD formulae: namely mono-2-O-, mono-3-O- and mono-6-O-.
Formula (XI) represents the alkenyl-CD di-derivatives, the derivatives being formed on the same glucoside unit or on different units (positions A-B, A-C, etc . . . ) with identical or different regioselectivities.
Compounds with general formulae (X) and (XI) can be grafted onto functionalised supports to produce supports with general formulae (Iy), (Ia), (Ib), (Iz), (Ic) and (Id), the vacant hydroxyl functions also may or may not be derived before or after grafting to the support. As an example, 3-mercaptopropyltrimethoxysilane was grafted to a silica gel via a covalent bond as follows: 
where R1=R2=CH3xe2x80x94CH2xe2x80x94Oxe2x80x94
and R3=xe2x80x94(CH2)3xe2x80x94
The reaction was carried out in xylene to eliminate firstly the water contained in the silica gel and then to eliminate the ethanol formed by hydrolysis of the ethoxysilane functions. The support obtained had the following characteristics:
%C=5.26
%S=2.68
%H=1.10
giving, on calculation, a thiol function density of 0.85 mmol/g, or 0.89 if it is considered that 2 or 3 ethoxysilanes have effectively reacted with the silica gel. 30% of the xe2x80x9ctheoreticalxe2x80x9d SiOH had been modified. Starting from a density of 8xc3x9710xe2x88x926 moles of SiOH/m2 with a support of 360 m2/g, we arrive at a density of 28 mmoles/g of silica. The support obtained, xe2x80x9cmercaptopropylsilicaxe2x80x9d or xe2x80x9cthiolxe2x80x9d silica, was then reacted with a compound with formula (X) or (XI). Anti-Markovnikov addition of the double bond to the silica thiol was carried out in the presence of a free radical initiator (C. Rosini, TETRAHEDRON LETT. 1985, 26 (28), 3361, and A. Tambute, NEW J. CHEM., 1989, 13, 625-637).
As an example, mono-2-O-pentenyl-xcex2-cyclodextrin was grafted to the thiol support as shown in the following reaction scheme: 
where R1=R2=CH3xe2x80x94CH2xe2x80x94Oxe2x80x94
R3=xe2x80x94(CH2)3xe2x80x94
R4=xe2x80x94(CH2)3xe2x80x94
and n=7.
The reaction was carried out in chloroform under reflux in the presence of azo-isobutyronitrile (AIBN). The supports obtained form part of the invention.
These supports could then be oxidised to transform the thioether function to a sulphoxide using hydrogen peroxide (xe2x80x9cOrganic compounds of bivalent sulphurxe2x80x9d vol. 2, pp. 64-66, Chemical Publishing Company, New York, 1960), indobenzene dichloride (Barbieri, J. CHEM. SOC. C659, 1968), sodium metaperiodate (Leonard, J. ORG. CHEM., 27, 282, 1962) or tertiobutyl oxychloride (Walling, J. ORG. CHEM. 32 1286, 1967) or peracids.
The sulphoxide supports obtained could be oxidised to sulphones using potassium permanganate or hydrogen peroxide (Heubert, CHEM. COMM., 1036, 1968 and Curci, TETRAHEDRON LETT., 1749, 1963). 
The preferred oxidising agent is hydrogen peroxide. The reaction solvent is generally water or an alcohol or an organic solvent which is miscible with water. The bulk temperature is 10xc2x0 C. to 40xc2x0 C. The reaction time is 1 to 8 hours.
As an example, the above support, a mono-2-O-pentyl-xcex2-cyclodextrin grafted onto thiol silica, was directly oxidised to the sulphone with excess hydrogen peroxide in solution in water/methanol at 25xc2x0 C. The reaction kinetics were monitored by following the hydrogen peroxide content in the reaction medium by quantitative analysis using a reducing agent.
The oxidised support, of type (Ib), had the following structure: 
where n=7
R3=R4=xe2x80x94(CH2)3xe2x80x94
R1=R2=CH3xe2x80x94CH2xe2x80x94Oxe2x80x94
or OH
or O-support
The performances of supports (Iy) and (Ib) were compared by separating enantiomers using high performance liquid chromatography (HPLC). The supports were compressed under 500 bars pressure in 250 mmxc3x974.6 mm HPLC tubes (length by internal diameter) under identical conditions.
kxe2x80x21 and kxe2x80x22 are partition ratios, i.e., when I=1 or 2, kxe2x80x21=(tR1xe2x88x92t0)/t0, where tR1 is the retention time of compound I and t0 is the dead time;
xcex1 is the relative retention ratio: xcex1=(tR2xe2x88x92t0)/(tR1xe2x88x92t0)=kxe2x80x22/kxe2x80x22;
RS is the peak resolution:             R      s        =                  1        4            ⁢              (                              α            -            1                    α                )            ⁢              (                              k            2            xe2x80x2                                1            +                          k              2              xe2x80x2                                      )            ⁢                        (          N          )                          1          /          2                      ,
where N is the number of plates;
N=a(tR/xcfx89)2 where xcfx89=the peak width at a given ordinate, proportional to the square of the standard deviation or variance "sgr"2 by the relationship xcfx892=xcex1"sgr"2;
giving N=16(tR/xcfx89)2=5.54(tR/xcfx89)2.
The results are shown in Table 1 on the next page.
There is an interest in modifying all or some of the hydroxyl functions of a cyclodextrin to modify the encapsulation properties thereof. Complete modification of the hydroxyl functions confers different properties on the cyclodextrin to those of native cyclodextrins. As an example, methylated cyclodextrins are more soluble than those obtained from native CDs.
The support for the preceding example was modified by the action of an isocyanate. Adding alcohols to isocynates is known per se (Satchell, CHEM. SOC. REV. 4, 231-250, 1975). The reaction is generally carried out in a basic medium (for example pyridine, triethylamine) in the presence of an organometallic catalyst (Davies, J. CHEM. SOC. C2663, 1967).
The bulk temperature is from 50xc2x0 C. to 150xc2x0 C. It is preferably kept between 80xc2x0 C. and 120xc2x0 C. bulk. The reaction period is between 1 and 48 hours and is adjusted as a function of the reactivity of the isocyanate used towards the alcohol to be transformed.
In the preceding support example, the primary and secondary alcohol functions, vacant in the xcex2-CD, were percarbamated.by 3,5-dimethylphenylisocyanate in a 50/50 triethylamine/pyridine mixture at 80xc2x0 C. bulk over 24 hours in, the presence of dibutylin laurate.
This support had the following structure: 
where n=7
R3=R4=xe2x80x94(CH2)3xe2x80x94
R1=R2=CH3xe2x80x94CH2xe2x80x94Oxe2x80x94
or OH
or O-support
and Z1=Z2=Z3=